Generation of inducible SMARCAL1 knock-down iPSC to model severe Schimke immune-osseous dysplasia reveals a link between replication stress and altered expression of master differentiation genes

ABSTRACTThe Schimke immuno-osseous dysplasia is an autosomal recessive genetic osteochondrodysplasia characterized by dysmorphism, spondyloepiphyseal dysplasia, nephrotic syndrome and frequently T cell immunodeficiency. Several hypotheses have been proposed to explain pathophysiology of the disease, however, the mechanism by which SMARCAL1 mutations cause the syndrome is elusive. Indeed, animal models of the disease are absent or useless to provide insight into the disease mechanism, since they do not recapitulate the phenotype. We generated a conditional knockdown model of SMARCAL1 in iPSCs to mimic conditions of cells with severe form the disease. Here, we characterize this model for the presence of phenotype linked to the replication caretaker role of SMARCAL1 using multiple cellular endpoints. Our data show that conditional knockdown of SMARCAL1 in human iPSCs induces replication-dependent and chronic accumulation of DNA damage triggering the DNA damage response. Furthermore, they indicate that accumulation of DNA damage and activation of the DNA damage response correlates with increased levels of R-loops and replication-transcription interference. Finally, we provide data showing that, in SMARCAL1-deficient iPSCs, DNA damage response can be maintained active also after differentiation, possibly contributing to the observed altered expression of a subset of germ layer-specific master genes. In conclusion, our conditional SMARCAL1 iPSCs may represent a powerful model where studying pathogenetic mechanisms of severe Schimke immuno-osseous dysplasia, thus overcoming the reported inability of different model systems to recapitulate the disease.

Download Full-text

Replication Associated Stress in the Fetal Stem Cell Pool of FA Mice Can be Rescued By Pharmacologic Inhibition of Constitutively Activated P38 MAPK

Blood ◽

10.1182/blood.v126.23.2411.2411 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2411-2411

Author(s):

Youngme Yoon ◽

Ashley N. Kamimae-Lanning ◽

Kelsie Storm ◽

Natalya A Goloviznina ◽

Peter Kurre

Keyword(s):

Dna Damage ◽

Stress Response ◽

P38 Mapk ◽

Dna Damage Response ◽

Colony Formation ◽

Model Systems ◽

Donor Chimerism ◽

Damage Response ◽

Physiologic Role

Abstract Fanconi Anemia (FA) is a rare, recessively heritable disorder with prominent failure of hematopoiesis. The physiologic role of FA proteins has not been fully resolved to date. While several existing model systems delineate its role in DNA damage response caused by alkylating agents, aldehydes, and inflammatory cytokines, all rely on experimental induction. We previously demonstrated the in utero onset of hematopoietic failure in mice with genetic disruption of Fancc. Herein, we found significant deficits in the fetal liver (FL) hematopoietic stem and progenitor cell (HSPC) pool in Fancd2 mice. Both AA4.1+ Sca-1+ Lin- expressing progenitors (ASL) and CD48- CD150+ Lin- Sca-1+ (SLAM) cells were decreased in frequency in Fancd2-/- versus WT FL. Similarly, we observed a significant decrease in progenitor colony formation and deficits in primary and secondary transplantation among Fancd2-/- FL compared to WT. Fancd2-/- FL cells were characteristically sensitive to mitomycin C and had significantly fewer SLAM cells in the G0 phase of cell cycle and elevated p21 expression, indicating canonical P53 activation. Consistent with prior reports by other groups on embryonic stem cells and our own Fancc-/- FL studies, we found neither exaggerated frequency of apoptotic cells, nor transcriptional induction of Puma or Noxa. We hypothesized that the observed deficits in developmental HSPC pool expansion reflect replication-associated stress. At the transcriptional level, we found activation of the DNA damage response via Rad51 and Prkdc, corroborated by immunofluorescent imaging of Rad51 foci as well as comet assays in FL cells. Next, we tested P38 MAPK as a stress response previously found to confer repopulation deficits in postnatal BM failure among Fancc and Fanca mice; here, our experiments revealed baseline (unprovoked) activation of phospho-p38 and rescue of Fancd2-/- progenitor colony formation using a pharmacological inhibitor, SB203580. Results were further strengthened by transplantation, revealing increased Fancd2-/- donor chimerism after in vivo administration of SB203580. The gains in donor chimerism persisted even after cessation of drug administration. These results suggest that replication-associated stress in the rapidly cycling fetal Fancd2-/- HSPC pool evokes a cellular stress response that constrains physiological expansion. Our work emphasizes the prenatal onset of hematopoietic failure and reveals pharmacological rescue by inhibition of constitutively active P38 MAPK. Furthermore, FA fetal hematopoiesis is an original model of unprovoked hematopoietic failure that allows the study of physiologic role of FA proteins in HSPC. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Characterization of the role of the DNA damage response pathway in parvoviral replication

10.32469/10355/15925 ◽

2012 ◽

Author(s):

Richard Adeyemi

Keyword(s):

Dna Damage ◽

Dna Damage Response ◽

Damage Response ◽

Dna Damage Response Pathway

Download Full-text

Faculty Opinions recommendation of A systems approach to delineate functions of paralogous transcription factors: role of the Yap family in the DNA damage response.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1103427.559478 ◽

2008 ◽

Author(s):

Jürg Bahler

Keyword(s):

Dna Damage ◽

Transcription Factors ◽

Dna Damage Response ◽

Systems Approach ◽

Damage Response

Download Full-text

DNA Damage Response in Multiple Myeloma: The Role of the Tumor Microenvironment

Cancers ◽

10.3390/cancers13030504 ◽

2021 ◽

Vol 13 (3) ◽

pp. 504

Author(s):

Takayuki Saitoh ◽

Tsukasa Oda

Keyword(s):

Multiple Myeloma ◽

Dna Damage ◽

Dna Repair ◽

Tumor Microenvironment ◽

Dna Damage Response ◽

Genetic Instability ◽

Dna Repair Mechanisms ◽

Damage Response ◽

Repair Mechanisms

Multiple myeloma (MM) is an incurable plasma cell malignancy characterized by genomic instability. MM cells present various forms of genetic instability, including chromosomal instability, microsatellite instability, and base-pair alterations, as well as changes in chromosome number. The tumor microenvironment and an abnormal DNA repair function affect genetic instability in this disease. In addition, states of the tumor microenvironment itself, such as inflammation and hypoxia, influence the DNA damage response, which includes DNA repair mechanisms, cell cycle checkpoints, and apoptotic pathways. Unrepaired DNA damage in tumor cells has been shown to exacerbate genomic instability and aberrant features that enable MM progression and drug resistance. This review provides an overview of the DNA repair pathways, with a special focus on their function in MM, and discusses the role of the tumor microenvironment in governing DNA repair mechanisms.

Download Full-text

Dissecting the Role of Thyrotropin in the DNA Damage Response in Human Thyrocytes after 131I, γ Radiation and H2O2

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/clinem/dgz185 ◽

2019 ◽

Vol 105 (3) ◽

pp. 839-853

Author(s):

Aglaia Kyrilli ◽

David Gacquer ◽

Vincent Detours ◽

Anne Lefort ◽

Frédéric Libert ◽

...

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Dna Damage ◽

Dna Damage Response ◽

Iodide Uptake ◽

Transcriptomic Response ◽

Uptake Inhibition ◽

Γ Radiation ◽

Damage Response

Abstract Background The early molecular events in human thyrocytes after 131I exposure have not yet been unravelled. Therefore, we investigated the role of TSH in the 131I-induced DNA damage response and gene expression in primary cultured human thyrocytes. Methods Following exposure of thyrocytes, in the presence or absence of TSH, to 131I (β radiation), γ radiation (3 Gy), and hydrogen peroxide (H2O2), we assessed DNA damage, proliferation, and cell-cycle status. We conducted RNA sequencing to profile gene expression after each type of exposure and evaluated the influence of TSH on each transcriptomic response. Results Overall, the thyrocyte responses following exposure to β or γ radiation and to H2O2 were similar. However, TSH increased 131I-induced DNA damage, an effect partially diminished after iodide uptake inhibition. Specifically, TSH increased the number of DNA double-strand breaks in nonexposed thyrocytes and thus predisposed them to greater damage following 131I exposure. This effect most likely occurred via Gα q cascade and a rise in intracellular reactive oxygen species (ROS) levels. β and γ radiation prolonged thyroid cell-cycle arrest to a similar extent without sign of apoptosis. The gene expression profiles of thyrocytes exposed to β/γ radiation or H2O2 were overlapping. Modulations in genes involved in inflammatory response, apoptosis, and proliferation were observed. TSH increased the number and intensity of modulation of differentially expressed genes after 131I exposure. Conclusions TSH specifically increased 131I-induced DNA damage probably via a rise in ROS levels and produced a more prominent transcriptomic response after exposure to 131I.

Download Full-text

The role of Schizosaccharomyces pombe Rad32, the Mre11 homologue, and other DNA damage response proteins in non-homologous end joining and telomere length maintenance

Nucleic Acids Research ◽

10.1093/nar/27.13.2655 ◽

1999 ◽

Vol 27 (13) ◽

pp. 2655-2661 ◽

Cited By ~ 50

Author(s):

S. Wilson ◽

N. Warr ◽

D. L. Taylor ◽

F. Z. Watts

Keyword(s):

Dna Damage ◽

Schizosaccharomyces Pombe ◽

Telomere Length ◽

Dna Damage Response ◽

End Joining ◽

Damage Response ◽

Non Homologous End Joining

Download Full-text

Role of H2AX in DNA damage response and human cancers

Mutation Research/Reviews in Mutation Research ◽

10.1016/j.mrrev.2008.08.003 ◽

2009 ◽

Vol 681 (2-3) ◽

pp. 180-188 ◽

Cited By ~ 53

Author(s):

Niloo Srivastava ◽

Sailesh Gochhait ◽

Peter de Boer ◽

Rameshwar N.K. Bamezai

Keyword(s):

Dna Damage ◽

Dna Damage Response ◽

Human Cancers ◽

Damage Response

Download Full-text

A novel role of the chromokinesin Kif4A in DNA damage response

Cell Cycle ◽

10.4161/cc.7.13.6130 ◽

2008 ◽

Vol 7 (13) ◽

pp. 2013-2020 ◽

Cited By ~ 37

Author(s):

Guikai Wu ◽

Longen Zhou ◽

Lily Khidr ◽

Xuning Emily Guo ◽

Wankee Kim ◽

...

Keyword(s):

Dna Damage ◽

Dna Damage Response ◽

Damage Response

Download Full-text

Nucleotide Excision Repair Protein Rad23 Regulates Cell Virulence Independent of Rad4 in Candida albicans

mSphere ◽

10.1128/msphere.00062-20 ◽

2020 ◽

Vol 5 (1) ◽

Cited By ~ 1

Author(s):

Jia Feng ◽

Shuangyan Yao ◽

Yansong Dong ◽

Jing Hu ◽

Malcolm Whiteway ◽

...

Keyword(s):

Dna Damage ◽

Candida Albicans ◽

Nucleotide Excision Repair ◽

Dna Damage Response ◽

Excision Repair ◽

Content Type ◽

Virulence Regulation ◽

Nucleotide Excision ◽

Damage Response

ABSTRACT In the pathogenic yeast Candida albicans, the DNA damage response contributes to pathogenicity by regulating cell morphology transitions and maintaining survival in response to DNA damage induced by reactive oxygen species (ROS) in host cells. However, the function of nucleotide excision repair (NER) in C. albicans has not been extensively investigated. To better understand the DNA damage response and its role in virulence, we studied the function of the Rad23 nucleotide excision repair protein in detail. The RAD23 deletion strain and overexpression strain both exhibit UV sensitivity, confirming the critical role of RAD23 in the nucleotide excision repair pathway. Genetic interaction assays revealed that the role of RAD23 in the UV response relies on RAD4 but is independent of RAD53, MMS22, and RAD18. RAD4 and RAD23 have similar roles in regulating cell morphogenesis and biofilm formation; however, only RAD23, but not RAD4, plays a negative role in virulence regulation in a mouse model. We found that the RAD23 deletion strain showed decreased survival in a Candida-macrophage interaction assay. Transcriptome sequencing (RNA-seq) and quantitative real-time PCR (qRT-PCR) data further revealed that RAD23, but not RAD4, regulates the transcription of a virulence factor, SUN41, suggesting a unique role of RAD23 in virulence regulation. Taking these observations together, our work reveals that the RAD23-related nucleotide excision pathway plays a critical role in the UV response but may not play a direct role in virulence. The virulence-related role of RAD23 may rely on the regulation of several virulence factors, which may give us further understanding about the linkage between DNA damage repair and virulence regulation in C. albicans. IMPORTANCE Candida albicans remains a significant threat to the lives of immunocompromised people. An understanding of the virulence and infection ability of C. albicans cells in the mammalian host may help with clinical treatment and drug discovery. The DNA damage response pathway is closely related to morphology regulation and virulence, as well as the ability to survive in host cells. In this study, we checked the role of the nucleotide excision repair (NER) pathway, the key repair system that functions to remove a large variety of DNA lesions such as those caused by UV light, but whose function has not been well studied in C. albicans. We found that Rad23, but not Rad4, plays a role in virulence that appears independent of the function of the NER pathway. Our research revealed that the NER pathway represented by Rad4/Rad23 may not play a direct role in virulence but that Rad23 may play a unique role in regulating the transcription of virulence genes that may contribute to the virulence of C. albicans.

Download Full-text